Pitx2 is the homeobox gene mutated in Rieger syndrome type I (RGS I) whose patients manifest tooth abnormalities including hypodontia, anodontia vera, abnormally shaped teeth and other craniofacial dysmorphologies. Importantly, RGS I results from haploinsufficiency revealing that one half dose of Pitx2 is insufficient for normal development. To uncover the complete function of Pitx2 in craniofacial development, we have generated a Pitx2 allelic series in mice. Our lab and others showed that Pitx2 was necessary for tooth morphogenesis to progress beyond the bud stage. In specific aims 1 and 2 of this application, we will test the hypothesis that Pitx2 functions as a downstream component of a ft catenin dependent, Wnt signaling pathway that is required for expansion of dental epithelium. In addition to tooth morphogenesis, RGS I patients have facial dysmorphologies such as midface hypoplasia. Using our allelic series, we have found that Pitx2 regulates craniofacial bone and muscle development. As suggested by our preliminary data, the hypothesis that Pitx2 autonomously regulates formation of first branchial arch-derived muscle will be tested in specific aim 3. Our recent observation that Pitx2 coordinately regulates the Fgf8 and Bmp4 signaling pathways forms that basis for specific aim 4. We will focus on the hypothesis that Pitx2 regulates craniofacial bone development through a mechanism that involves the F/fce2-regulated Bmp4 repression pathway. The experiments proposed here, investigating the genetic pathways upstream and downstream of Pitx2, will provide insight into fundamentally important questions of craniofacial development that are relevant to human disease.